CN104232600A - Preparation of glyphosate resistance enhanced EPSPS (5-enolpyruvyl shikimate-3-phosphate synthase) mutant of Vitis vinifera and application of EPSPS mutant of Vitis vinifera - Google Patents
Preparation of glyphosate resistance enhanced EPSPS (5-enolpyruvyl shikimate-3-phosphate synthase) mutant of Vitis vinifera and application of EPSPS mutant of Vitis vinifera Download PDFInfo
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Abstract
The invention discloses a glyphosate resistance enhanced EPSP (5-enolpyruvyl shikimate-3-phosphate synthase) synthase multi-site mutant which is derived from Vitis vinifera and prepared by adopting a DNA shuffling technology, as well as a coding gene and application of the mutant. The mutant comprises 7 mutation sites, wherein the amino acid sequence of the mutant is as shown in the SEQ ID No.1, and the base sequence coded by the mutant is as shown in the SEQ ID No.2. Tests indicate that the EPSP synthase multi-site mutant derived from Vitis vinifera and the gene coded by the mutant have relatively high glyphosate resistance and relatively strong PEP affinity, and the characteristics of the mutant and the gene provide possibility to the gene for culturing anti-glyphosate transgenic crops.
Description
Technical field
The invention belongs to microorganism field, relate to one derive from grape (
vitis vinifera) the preparation of 5-enolpyruvylshikimate-3-phosphate synthase (EPSP synthase/EPSPS) multisite mutant and encoding gene and application, be specifically related to one utilize DNA molecular to reset (DNA Shuffling) and functional complementation to carry out transformation acquisition multisite mutant to deriving from grape epsp synthase gene, the gene of then being encoded is applied in the Resistence research of glyphosate and rice conversion, genetically modified crops cultivation.
Background technology
Shikimic acid pathway is the important channel of plant and the synthesis of microorganism die aromatischen Aminosaeuren.EPSP synthase is the key enzyme of shikimic acid pathway, catalysis 3-phosphoric acid shikimic acid (S3P) and phosphoenolpyruvic acid (PEP) generate 5-enolpyruvyl-shikimate-3-phosphate synthase (5-enolpyruvyl shikimate-3-phosphate synthase, EPSPS).Herbicide glyphosate is the analog of PEP, can with the activity of PEP competitive inhibition EPSPS, thus block the biosynthesizing of die aromatischen Aminosaeuren, finally cause Plant death.Up to now, the gene being used successfully to business-like resistance glyphosate genetically modified crops only has the EPSPS gene of the Agrobacterium CP4 of II type.But because this gene source is in bacterium, thus easily cause the psychology of ordinary consumer to scruple.Have scholar to report recently to derive from the EPSPS gene of edible plants in genetically modified crops, show good glyphosate resistance (Plant Physiol 140:184-195), namely this just develop resistance glyphosate genetically modified crops for development resistance glyphosate genetically modified crops provide a kind of new approach based on the EPSPS gene of plant-sourced comes.But the wild-type EPSPS gene deriving from plant is often very sensitive to glyphosate, thus can not be directly used in the cultivation of genetically modified crops.This just needs to be transformed it by genetic engineering means, thus obtains the good novel gene that can be used for genetically modified crops cultivation of the novel function deriving from plant.
Summary of the invention
Technical problem to be solved by this invention be to provide a kind of derive from grape EPSP synthase multisite mutant and encoding gene and application.Reset (DNA Shuffling) by adopting DNA molecular and obtain this EPSP synthase multisite mutant library, then having complementary functions of intestinal bacteria aroA mutant strain (ER2799) is utilized to screen the EPSP synthase mutant library deriving from grape, obtain a multisite mutant containing 7 amino acid mutations, its aminoacid sequence is as shown in SEQ ID No.1.Through verification experimental verification, this mutant not only shows high glyphosate resistance significantly in intestinal bacteria, and also shows stable glyphosate resistance in transgenic plant.
In order to reach above object, the present invention realizes by the following technical solutions:
First, applicant inputs 5-enolpyruvylshikimate-3-phosphate synthase and Vitis vinifera at NCBI website (http://www.ncbi.nlm.nih.gov), obtains the mRNA sequence that sequence number is the coding grape 5-enol pyruvoyl-shikimic acid-3-phosphoric acid synthetase gene of NM_001281247.According to this sequences Design one couple of PCR primers (VvEPSPSZ and VvEPSPSF), with the kyoto grape cDNA library (northwest Botany Gazette, 2009,29:1723-1729) of this test preservation for template, RT-PCR amplification acquisition grape epsp synthase gene (
vvEPSPS) full-length cDNA sequence.
Utilize sepharose to reclaim test kit to reclaim
vvEPSPSgene fragment, with DNase I damping fluid (50mmol/L Tris-Cl pH7.4+1mmol/L MgCl
2) 100 μ l dissolve; Add 0.1U DNase I, 25 DEG C process 15 minutes.70 DEG C process 10 minutes.10% acrylamide gel electrophoresis, saturating suction bag method reclaims the DNA small segment of 10 ~ 50bp.Carry out Primerless pcr amplification, reaction system: 5 μ l small pieces segment DNA+4 μ l 2.5mmol/L dNTPs+4.5 μ l 25mmol/L MgCl
2+ Taq2U+ddH
2o to 50 μ l; Response procedures is: 94 DEG C of 30s, 40 DEG C of 30s, 72 DEG C of 30s, totally 45 circulations.
With without primer PCR amplified production for template, with VvEPSPSZ and VvEPSPSF for primer carries out pcr amplification.Reaction system: 5 μ l Primerless PCR primer+VvEPSPSZ 0.2ng+VvEPSPSF 0.2ng+10 × PCR Buffer 5 μ l+2.5mmol/L dNTPs 4 μ l+Taq2U+ddH
2o to 50 μ l.Response procedures is: 94 DEG C of 30s, 70 DEG C of 30s, 72 DEG C of 2.0 min, totally 35 circulations, and recovery is reset
vvEPSPSgene fragment.
By the rearrangement of above-mentioned recovery
vvEPSPSgene fragment, after BamH I and Sac I double digestion, is built into prokaryotic expression carrier pG251(CN1338515) between promotor and t1t2 terminator, this carrier is with ampicillin resistance gene.Electric shocking method transform Escherichia coli strain DH5 α obtains mutant expression library, and storage capacity reaches 10
8, then extract test kit (Omega company of the U.S.) in a large number with plasmid and carry out plasmid extraction.Get the plasmid that 1 μ l extracts in a large number and proceed to intestinal bacteria ER2799(NEB company) coating cultivates 48h with containing on the M9 flat board of 200mM glyphosate, well-grown colony inoculation is cultivated to the M9 flat board containing 200mM glyphosate, find to only have 1 clone can containing the M9 grow on plates of 200mM glyphosate, the plasmid called after pVvEPSPS contained by it
mutant.
Utilize the glyphosate highly-tolerant plasmid pVvEPSPS that progressively sequencing method obtains above-mentioned screening
mutantcomplete sequence carries out DNA sequencing.Sequencing result shows this mutant on the same molecule containing following 7 mutational sites, respectively: mutational site 1(Q93R), the glutamine namely in EPSP synthase aminoacid sequence on the 93rd is replaced by arginine; Mutational site 2(T113A), the Threonine namely on the 113rd is replaced by L-Ala; Mutational site 3(P117L), namely the proline(Pro) of the 117th replaces with leucine; Mutational site 4(G126A), the glycine namely on the 126th is replaced by L-Ala; Mutational site 5(C160Y), the halfcystine namely on the 160th is replaced by tyrosine; Mutational site 6(N239H), the l-asparagine namely on the 239th is replaced by Histidine; Mutational site 7(V343A), the α-amino-isovaleric acid namely on the 343rd is replaced by L-Ala.Its aminoacid sequence of above-mentioned obtained mutant is as shown in SEQ ID No.1, and the base sequence of coding is as shown in SEQ ID No.2.
By glyphosate highly-tolerant plasmid pVvEPSPS obtained above
mutantproceed to paddy rice, tested by rice seed germination and Glyphosate Spray Tests illustrate of the present invention derive from grape EPSP synthase multisite mutant and coding gene (
vvEPSPS mutant ), not only have higher glyphosate resistance but also remain the affinity stronger with PEP, the cultivation being used for genetically modified crops for this gene is provided possibility by these characteristics.
Term of the present invention is identical with its universal.
Described " Nucleotide " and " primer " sequence is 5 ' end to 3 ' end.
Described " biomass cells " refers to microorganism, vegetable cell or tissue.
Described " microorganism " refers to prokaryotic micro-organisms or eukaryotic microorganisms, and prokaryotic micro-organisms is mainly bacterium.
Accompanying drawing explanation
Fig. 1 is the external glyphosate resistance test of high glyphosate resistant EPSP synthase mutant.
vvePSPS
matant for the high glyphosate resistant EPSP synthase mutant that the present invention obtains,
vvePSPS is grape wild-type EPSP synthase.
Fig. 2 for turn an encode EPSP synthase multisite mutant of the present invention (
vvePSPS
mutant) paddy rice of gene is containing the sprouting lab diagram on different concns glyphosate flat board, wherein Mur is the different rice strains turning encode EPSP synthase multisite mutant gene of the present invention, WTr turns the paddy rice that wild-type derives from the epsp synthase gene of grape, and CK is contrast.
Fig. 3 for turn an encode EPSP synthase mutant of the present invention (
vvePSPS
mutant) paddy rice 1.0 %(v/v of gene) Roundup sprays process phenotypic map, wherein Mur1 is the rice strain turning encode EPSP synthase multisite mutant gene of the present invention, WTr1 turns the paddy rice that wild-type derives from the epsp synthase gene of grape, and CK is contrast.
Embodiment
The DNA molecular of embodiment 1 epsp synthase gene resets (DNA Shuffling)
The 1.1 resistance glyphosate herbicide resistance gene deriving from grape
vvEPSPSsynthesis
Applicant inputs 5-enolpyruvylshikimate-3-phosphate synthase and Vitis vinifera at NCBI website (http://www.ncbi.nlm.nih.gov), obtains the mRNA sequence that sequence number is the coding grape 5-enol pyruvoyl-shikimic acid-3-phosphoric acid synthetase gene of NM_001281247.According to this sequences Design one couple of PCR primers (VvEPSPSZ:5'-AAGGATCCATGGCCTCTGTCGCCACTAAG
-3' and VvEPSPSF:5'-AAGAGCTCTCAATGTTTGGTAAAACGCTGG-3'), with kyoto grape cDNA library (the northwest Botany Gazette that this test is preserved, 2009,29:1723-1729) be template, RT-PCR amplification acquisition grape epsp synthase gene (
vvePSPS) full-length cDNA sequence.Reaction system: 1 μ lcDNA+4 μ l 2.5mmol/L dNTPs+25 μ l Buffer+KOD Plus(Toyobo Japan) polysaccharase 1U+1 μ l VvEPSPSZ+1 μ l VvEPSPSF+ddH
2o to 50 μ l; Response procedures is: 94 DEG C of 30s, 54 DEG C of 30s, 72 DEG C of 120s, totally 45 circulations), 2% Agrose electrophoresis detection pcr amplification result.
After PCR terminates, 1%(w/v) agarose gel recovery, adopt TArget Clone-Plus-test kit (Toyobo Japan) to connect.In transformation of E. coli DH5 α competent cell, acquisition contains
vvEPSPSthe positive colony of gene, extracting plasmid, order-checking.Namely obtain wild-type grape epsp synthase gene (
vvEPSPS).
1.2 pcr amplification epsp synthase gene and recovery
Contain with above-mentioned obtained
vvEPSPSthe positive plasmid of gene is template, VvEPSPSZ and VvEPSPSF is primer amplification epsp synthase gene, reaction conditions is: 94 DEG C of 10min denaturations, 94 DEG C of sex change 30s, 72 DEG C of annealing and extension 1.5min, totally 30 circulations, 1% Agrose electrophoresis, saturating suction bag method reclaims to obtain the epsp synthase gene fragment of 1368bp.
1) DNase I degradation of dna and recovery small segment
The epsp synthase gene fragment of above-mentioned recovery is with DNase I damping fluid (50mmol/L Tris-Cl pH7.4+1mmol/L MgCl
2) 100 μ l dissolve; Add 0.1U DNase I, 25 DEG C process 15 minutes.70 DEG C process 10 minutes.10% acryl amide electrophoresis, saturating suction bag method reclaims the small segment of 10 ~ 50bp.With 10 μ l 10 × without primer PCR damping fluid (Primerless PCR Buffer) (50mmol/L KCl+10mmol/L Tris-Cl pH9.0+1% Triton) dissolution precipitation.
2) without primer PCR (Primerless PCR)
Carry out Primerless pcr amplification.Reaction system: 5 μ l small pieces segment DNA+4 μ l 2.5mmol/L dNTPs+4.5 μ l 25mmol/L MgCl
2+ Taq2U+ddH
2o to 50 μ l; Response procedures is: 94 DEG C of 30s, 40 DEG C of 30s, 72 DEG C of 30s, totally 45 circulations), 2% Agrose electrophoresis detection pcr amplification result.
3) primer PCR (Primer PCR) is had
With above-mentioned without primer PCR amplified production for template, with VvEPSPSZ and VvEPSPSF for primer carries out PrimerPCR amplified reaction.Reaction system: 5 μ l Primerless PCR primer+VvEPSPSZ 0.2ng+VvEPSPSF 0.2ng+10 × PCR Buffer 5 μ l+2.5mmol/L dNTPs 4 μ l+Taq2U+ddH
2o to 50 μ l.Response procedures is: 94 DEG C of 30s, 70 DEG C of 30s, 72 DEG C of 2.0min, totally 35 circulations, 1%Agrose electrophoresis detection, reclaim 1368bp reset epsp synthase gene fragment.
Embodiment 2 high glyphosate resistant EPSP synthase screening mutant and sequential analysis
The screening of 2.1 high glyphosate resistant EPSP synthase mutant
The epsp synthase gene fragment of above-mentioned recovery being reset, after BamH I and Sac I double digestion, is built into prokaryotic expression carrier pG251(CN1338515) between promotor and t1t2 terminator, this carrier is with ampicillin resistance gene.Electric shocking method transform Escherichia coli strain DH5 α obtains mutant expression library, and storage capacity reaches 10
8, then extract test kit (Omega company of the U.S.) in a large number with plasmid and carry out plasmid extraction.
Get the plasmid that 1 μ l extracts in a large number and proceed to intestinal bacteria ER2799(NEB company) coating cultivates 48h with containing on the M9 flat board of 200mM glyphosate, finds that there is a colony growth good.By the plasmid (pVvEPSPS of this clone's extracting
mutant) again proceed to intestinal bacteria ER2799(NEB company) in, transformant sterile toothpick point is checked resistance with containing on the M9 solid medium of 200mM glyphosate, result proves that this is cloned the transformant produced and has resistance glyphosate characteristic, shows that resistance glyphosate characteristic is owing to having proceeded to this pVvEPSPS really
mutantplasmid causes.
The sequential analysis of the EPSP synthase mutant of 2.2 glyphosate highly-tolerants
Progressively sequencing methods is utilized to screen to 2.1 the glyphosate highly-tolerant plasmid pVvEPSPS obtained
mutantcomplete sequence carries out DNA sequencing.Analytical results shows, this high-resistance glyphosate mutant contains following 7 mutational sites on the same molecule, specifically: mutational site 1(Q93R), the glutamine namely in EPSP synthase aminoacid sequence on the 93rd is replaced by arginine; Mutational site 2(T113A), the Threonine namely on the 113rd is replaced by L-Ala; Mutational site 3(P117L), namely the proline(Pro) of the 117th replaces with leucine; Mutational site 4(G126A), the glycine namely on the 126th is replaced by L-Ala; Mutational site 5(C160Y), the halfcystine namely on the 160th is replaced by tyrosine; Mutational site 6(N239H), the l-asparagine namely on the 239th is replaced by Histidine; Mutational site 7(V343A), the α-amino-isovaleric acid namely on the 343rd is replaced by L-Ala.Its aminoacid sequence of above-mentioned obtained mutant is as shown in SEQ ID No.1, and the base sequence of coding is as shown in SEQ ID No.2.
The external glyphosate resistance test of embodiment 3
By the wild-type that embodiment 1 obtains
vvEPSPSgene, with after BamH I and Sac I double digestion, is built into prokaryotic expression carrier pG251 and obtains plasmid pVvEPSPS.The pVvEPSPS that pVvEPSPS plasmid and embodiment 2 obtain is transformed respectively with intestinal bacteria ER2799 bacterial strain
mutantplasmid, coats on M9 flat board and cultivates 48h, with toothpick picking separately transformant be inoculated in LB liquid nutrient medium and cultivate, treat that concentration reaches 5 × 10
3during cells/ μ L, get the enchylema point sample of 2 μ L separately after enchylema and 1/5 and 1/25 dilution respectively on the M9 flat board containing different concns glyphosate (0,50mM, 200mM), observe 50mM glyphosate and substantially inhibit wild-type after cultivation 48h
vvEPSPSthe growth of cell.But when glyphosate concentration is 200mM, mutant VvEPSPS
mutantcell then still can be bred.Mutant VvEPSPS as can be seen here
mutantrelative to wild-type VvEPSPS, have higher external glyphosate tolerant, result is referring to Fig. 1.
The prokaryotic expression of the EPSP synthase mutant of the high glyphosate resistance of embodiment 3
3.1 expression of EPSP synthase multisite mutant gene in intestinal bacteria
The EPSP synthase multisite mutant gene obtained with above-mentioned screening is masterplate, carries out pcr amplification with primer VvEPSPSZ and VvEPSPSF, with KOD Plus(Toyobo Japan) for Taq archaeal dna polymerase, amplification condition is followed successively by: 94 DEG C of 30s, 55 DEG C of 30s, 72 DEG C of 90s, 30 circulations of increasing.After loop ends, add the rtaq enzyme (Dalian Bao Bio-Engineering Company) of 2U, 72 DEG C extend 90s, the long 1368bp of amplified fragments.After PCR primer BamH I and Sac I enzyme are cut, be connected into the carrier pET-28a(NEB company that same enzyme is cut) obtain recombinant plasmid pET-VvEPSPS
mutantand by its transformation of E. coli BL21(DE3) (Novagen company), transformant is coated in LB solid medium and cultivates 24h.With gel-protein purification test kit HisTrap HP(Amersham Biosciences company) protein expression and purification is carried out to it, SDS-PAGE electrophoresis detection.Through SDS-PAGE electrophoresis detection, EPSP synthase multisite mutant gene (VvEPSPS of the present invention
mutant) the EPSP synthase mutant (VvEPSPS that encodes
mutant) albumen size is about 50kDa, conform to predictor.
Embodiment 4 VvEPSPS
mutantenzyme activity determination and Determination of Kinetic Parameters
4.1 measuring method
Inorganic phosphorus typical curve: 10 mM inorganic phosphorus are by 1: 10 dilution, get 0,1,2,3 respectively ... in 20 μ l and 1.5ml Eppendorf centrifuge tube, add pure water to 100 μ l to mix, add MAT solution (0.045% malachite green: 4.2% ammonium molybdate=3:1, V/V) 0.8ml mixing, timing adds SC solution (34% trisodium citrate) 100 μ l and mixes rapidly after three minutes, room temperature measures OD after leaving standstill 20min
660value.In triplicate.Take inorganic phosphorus concentration as X-coordinate, OD
660value is mapped for ordinate zou and is obtained inorganic phosphorus typical curve.
1, enzyme activity determination: protein quantification adopts Coomassie brilliant G-250 staining (Bradford, 1976).Following solution is added: 10mM PEP solution 2 μ l, 10 mM S3P solution 2 μ l, 0.5M HEPES solution 2 μ l, 1mM (NH on ice with 1.5ml Eppendorf centrifuge tube
4)
6mO
7o
244H
2o solution 2 μ l and distilled water 12 μ l mixes, after bathing 5min with 28 DEG C of temperature, each pipe sample room adds 5 μ l purifying proteins and timing every 2s, after 2min, interval 2s adds 200 μ l MAT solution successively again, after colour developing 3min, interval 2s adds 20 μ l 34% SC solution successively and mixes rapidly again, measures OD after color development at room temperature 20min
660value.Contrast except not adding except purifying protein, all the other same sample hoses.The OD of sample hose and control tube
660after value is subtracted each other, contrast inorganic phosphorus typical curve can try to achieve the inorganic phosphorus molar weight of reacting and discharging, more just obtains the enzyme activity of enzyme divided by reaction times and zymoprotein amount.
2, half amount of suppression (IC
50) measure: add 0,10 in above-mentioned reaction solution
-3, 10
-2, 10
-1, 1,10,100,500mM glyphosate, gained specific activity of enzyme data take glyphosate concentration as X-axis, adopt logarithmic coordinates, with speed of response (nkat/mg) for Y-axis mapping.
3, Km(PEP) measure: S3P strength of solution is constant at 1mM, different PEP concentration (0.05,0.067,0.1,0.2,0.5,1.0mM) under measure enzyme reaction rate by above-mentioned reaction system, survey numerical value and map by V-v/ [S] (Eadic-Hofstee) method.
4, Ki(glyphosate) measure: measure under different glyphosate concentration (0,10,50,100 μM) PEP concentration be 0.05,0.067,0.1,0.2,0.5,1.0mM time EPSP enzyme reaction rate.Take double-reciprocal plot, obtain 1/V-1/ [S] straight line, then using the slope of each straight line as ordinate zou, the concentration of glyphosate obtains a new straight line as X-coordinate, and the intersection point of this straight line and X-axis is Ki(glyphosate) value.
4.2 measurement result
VvEPSPS of the present invention
mutantenzyme kinetics parameter as shown in table 1 below.
The kinetic parameter of table 1 EPSP synthase multisite mutant
According to VvEPSPS
mutantenzyme kinetics parameter known, VvEPSPS of the present invention
mutantnot only have higher glyphosate resistance, but also remain the affinity stronger with PEP, these characteristics will be this VvEPSPS
mutantthe cultivation that synthase multisite mutant gene is used for genetically modified crops provides possibility.
The conversion of embodiment 5 paddy rice and the test of glyphosate resistance thereof
The acquisition of 5.1 transgenic paddy rices
1, the preparation of Agrobacterium
1) the single bacterium of picking Agrobacterium is inoculated in 5mL LB liquid nutrient medium (Rifampin 50 μ g/mL, paraxin 100 μ g/mL), 28 DEG C, cultivates 20h for 250 revs/min.
2) get 1mL bacterium liquid to transfer in 20 ~ 30mL LB liquid nutrient medium (Rifampin 50 μ g/mL, paraxin 100 μ g/mL), 28 DEG C, cultivate about 12h for 250 revs/min, survey OD
600≈ 1.5.
3) 8000 revs/min, 4 DEG C, 10min collected by centrifugation thalline, is resuspended in Agrobacterium-mediated Transformation penetrating fluid (5wt% sucrose, 0.05wt% Silwet L-77) and is diluted to OD
600≈ 0.8.
2, the nascent callus that Agrobacterium is infected and the embryo callus of being originated by the preculture mature embryo of 4 days with the Dual culture of Rice Callus or the immature embryo of cultivating 4 ~ 5d are originated immerses in ready agrobacterium suspension immediately, after infecting 30min, then callus is absorbed on aseptic filter paper unnecessary bacterium liquid, directly proceed to Dual culture and cultivate based on cultivation 3 ~ 4d under 23 DEG C of dark conditions.
3, the screening of resistant calli
Produced by the callus of Dual culture, rinsed with sterile water 3 ~ 4 times, then blots excessive moisture with aseptic filter paper, is proceeded to by callus on Selective agar medium, 28 DEG C of light culture, and two weeks subcultures once.
4, plant regeneration
After the screening of 2 ~ 3 generations, the vigorous resistant calli of growth selection is transferred on pre-division culture medium and is broken up process in advance; Resistant calli is transferred to division culture medium and (is broken up under 16h illumination every day, 8h dark, 28 DEG C of conditions by light culture again after 5 ~ 7 days, the seedling of regeneration cuts off original, strong plantlets and rootage on root media, move into phytotron subsequently potted plant, namely transgenic paddy rice is obtained, within initial several days, keep humidity, follow-up cultivation management conventionally carries out.
The Seed Germination Test of 5.2 transgenic paddy rices
By T1 for transposon mutant body VvEPSPS
mutant5 strains (Mur1-Mur5) of gene, first 2 strains (WTr1 and WTr2) of wild-type VvEPSPS and CK seed carry out surface sterilization with the alcohol-pickled 1min of 75%, then the NaClO solution of 2% active chlorine content (adding 1 ~ 3 Tween 20) is used to soak more than 60min (the longest can to 2h), and frequently shake, then use aseptic water washing 4 ~ 5 times.Next be evenly distributed in by seed on the 1/2MS flat board containing different concns glyphosate (0,500uM, 1000uM), Parafilm film seals, and is placed on 25 ° of C in thermostatic chamber, and 16 h light cultivate the sprouting situation that 6 days observe seed, and result is referring to Fig. 2.
Test finds: CK contrast and the seed turning wild-type VvEPSPS gene grows when glyphosate concentration is 500 μMs and be subject to serious suppression, and turn VvEPSPS of the present invention
mutantseed (Mur1, Mur3 and the Mur5) well-grown of mutant gene, and be 1000 μMs of transposon mutant body VvEPSPS at glyphosate concentration
mutantthe strain Mur1 of gene and Mur5 still well-grown, and form is normal.
(3) Glyphosate Spray Tests of transgenic paddy rice
By T3 for transposon mutant body VvEPSPS
mutantthe strain 1(Mur1 of gene) and the strain (WTr1) of wild-type VvEPSPS and CK contrast seed and first carry out surface sterilization with the alcohol-pickled 1min of 75%, then the NaClO solution of 2% active chlorine content (adding 1 ~ 3 Tween 20) is used to soak more than 60min (the longest can to 2h), and frequently shake, then use aseptic water washing 4 ~ 5 times.Next seed is wrapped on the plate that to be immersed in gauze containing moisture, 37 DEG C, cultivate 4 days, be transplanted to after seed shows money or valuables one carries unintentionally in nutrition platinum, be placed on 25 ° of C in thermostatic chamber, 16 h light cultivations.1.0%(v/v when little young plant grows to 12 ~ 15 centimetres high) Roundup sprays, and observe the growing state of young plant, result is referring to Fig. 3.
Test finds: with Roundup process after 10 days, and contrast and wild-type WTr1 young plant have started to wither, and turns VvEPSPS of the present invention
mutantthe young plant then well-grown of EPSP synthase mutant gene, and form is normal.
Attached mother liquor and each culture medium prescription:
One, mother liquor (stock solution) formula
1, MS
maxmother liquor (stock solution) (10X)
NH
4NO
3 16.5g
KNO
3 19.0g
MgSO
4·7H
2O 3.7g
CaCl
2·2H
2O
4.4g
Add water and be settled to 1000ml.
2, MS
minmother liquor (stock solution) (100X)
KI
0.083g
H
3BO
40.62g
MnSO
4·2H
2O 2.23g
ZnSO
4·7H
2O
0.86g
Na
2MoO
4·2H
2O
0.025g
CuSO
4·5H
2O
0.0025g
CoCl
2·2H
2O
0.0025g
Add water and be settled to 1000ml.
3, N6
maxmother liquor (stock solution) (10X)
KNO
3 28.3g
KH
2PO
4 4.0g
(NH
4)
2·SO
4 4.63g
MgSO
4·7H
2O
1.85g
CaCl
2·2H
2O 1.66g
Add water and be settled to 1000ml.
4, N6
minmother liquor (stock solution) (100X)
KI 0.08g
H
3BO
40.16g
MnSO
4·2H
2O
0.44g
ZnSO
4·7H
2O
0.15g
Add water constant volume to 1000ml
5, Fe
2+-EDTA mother liquor (100X)
FeSO
4·7H
2O
2.78g
Na
2EDTA·2H
2O
3.73g
Independent dissolving, then mixes, and adds water and is settled to 1000ml.
6, vitamin stock solution (Vitamin stock solution) (100X)
Nicotinic acid (Nicotinic acid) 0.1g
Vitamin B6 (Pyridoxine HCl, VB6) 0.1g
VITMAIN B1 (Thiaminc HCl, vb1) 0.1g
Glycine (Glycine) 0.2g
Inose (Inositol) 10g
Add water and be settled to 1000ml.
Two, culture medium prescription
1, Dual culture substratum
N6
maxmother liquor (N6
maxstock solution) (10X)
12.5ml
N6
minmother liquor (N6
minstock solution) (100X)
1.25ml
Fe
2+-EDTA mother liquor (Fe
2+-EDTA stock solution) (100X) 2.5ml
Vitamin stock solution (Vitamin stock solution) (100X)
2.5ml
Dichlorphenoxyacetic acid 2g/L(2,4-D) 0.75ml
Enzymic hydrolysis casein (Casein Enzymatic Hydrolysate)
0.2g
Sucrose (Sucrose) 5g
Agar powder (Agarose)
1.75g
Adding water to 250ml adjusts the front microwave oven thawing of pH=5.6 to add 5ml 50% glucose and 250 μ l 20g/L Syringylethanones.
2, Selective agar medium
N6
maxmother liquor (N6
maxstock solution) (10X) 25ml
N6
minmother liquor (N6
minstock solution) (100X)
2.5ml
Fe
2+-EDTA mother liquor (Fe
2+-EDTA stock solution) (100X)
2.5ml
Vitamin stock solution (Vitamin stock solution) (100X)
2.5ml
Dichlorphenoxyacetic acid 2g/L(2,4-D)
0.625ml
Enzymic hydrolysis casein (Casein Enzymatic Hydrolysate) 0.15g
Sucrose (Sucrose)
7.5g
Agar powder (Agarose) 1.75g
Add water to 250ml and adjust pH=6.0, add Totomycin and carboxylic benzyl with front thawing.
3, pre-division culture medium
MS
maxmother liquor (MS
maxstock solution) (10X) 25ml
MS
minmother liquor (MS
minstock solution) (100X) 2.5ml
Fe
2+-EDTA mother liquor (Fe
2+-EDTA stock solution) (100X)
2.5ml
Vitamin stock solution (Vitamin stock solution) (100X)
2.5ml
6-benzamido group purine 2g/L (6-BA)
0.5ml
Kinetin 2g/L(KT)
0.5ml
Indolylacetic acid 1mg/ml(IAA)
50 μ l
Enzymic hydrolysis casein (Casein Enzymatic Hydrolysate) 0.15g
Sucrose (Sucrose)
7.5g
Agar powder (Agarose) 1.75g
Add water to 250m and adjust pH=5.9, add Totomycin and carboxylic benzyl with front thawing.
4, division culture medium
MS
maxmother liquor (MS
maxstock solution) (10X)
100ml
MS
minmother liquor (MS
minstock solution) (100X) 10ml
Fe
2+-EDTA mother liquor (Fe
2+-EDTA stock solution) (100X)
10ml
Vitamin stock solution (Vitamin stock solution) (100X)
10ml
6-benzamido group purine 2g/L (6-BA)
2.0ml
Kinetin 2g/L(KT)
2.0ml
Indolylacetic acid 1mg/ml(IAA)
0.2ml
Naphthylacetic acid 1g/L (NAA) 0.2ml
Enzymic hydrolysis casein (Casein Enzymatic Hydrolysate) 1g
Sucrose (Sucrose)
30g
Plant gel (Phytagel)
3g
Add water to 1000ml and adjust pH=6.0, packing bottle.
5, root media
MS
maxmother liquor (MS
maxstock solution) (10X)
50ml
MS
minmother liquor (MS
minstock solution) (100X)
5ml
Fe
2+-EDTA （Fe
2+-EDTA stock solution) (100X) 10ml
Vitamin stock solution (Vitamin stock solution) (100X)
10ml
Agar powder (Sucrose) 20g
Plant gel (Phytagel) 3g
Add water to 1000ml and adjust pH=5.8, packing bottle.
<110> Academy of Agricultural Sciences, Shanghai City
<120> derives from the EPSP synthase multisite mutant of grape and the gene of coding thereof and application
<160> 2
<170> PatentIn version 3.3
<210> SEQ ID No 1
<211> 455
<212> PRT
<213> Vitis vinifera
MASVATKEKP STAPEIVLQP IKEISGTITL PGSKSLSNRI LLLAALSEGT TVVDNLLNSE 60
DVHYMLGALR TLGLHVEEQS ENKRVIVQGC GGRFPAGNGS VGEVQLFLGN AGAAMRLLTA 120
AVTAAAGNAS YVLDGVPRMR ERPIGDLVTG LKQLGADVNY FLGTNCPPVR VNGNGGLPGG 180
KVKLSGSISS QYLTALLMAA PLALGDVEIE IIDKLISIPY VEMTLKLMER FGVSVEHSHT 240
WDRFLIRGGQ KYKSPGNAFV EGDASSASYF LAGAAVTGGT VTVEGCGTSS LQGDVKFAEV 300
LEQMGAKVSW MENSVTVTGP PRDSSGRKHL RAIDVNMNKM PDAAMTLAVV ALYADGPTAI 360
RDVASWRVKE TERMIAICTE LRKLGATVEE GPDYCVITPP EKLNVTSIDT YDDHRMAMAF 420
SLAACADVPV TIKDPGCTRK TFPDYFEVLQ RFTKH* 455
<210> SEQ ID No 2
<211> 1368
<212> DNA
<213> Vitis vinifera
atggcctctg tcgccactaa ggagaagccg tcgacggcgc cggagattgt cctgcaaccc 60
attaaagaga tctccggcac catcacgttg ccgggttcca agtccctttc caatcggatc 120
ctgcttctag ctgctctctc tgagggaact actgttgtgg acaatttgtt aaatagtgag 180
gatgtccatt acatgctcgg agcactgagg acccttgggc tacatgtgga agagcaaagt 240
gaaaataaaa gagttattgt gcaaggttgt gggggccgat ttccagcggg aaatggatcg 300
gtaggtgaag tccaactttt cctaggaaat gctggagcag caatgcgtct attgacagcc 360
gcagttacag ctgctgctgg aaatgcaagc tatgtacttg atggggtgcc gcgcatgaga 420
gaaagaccaa ttggggatct agtcacaggt cttaagcagc tcggtgcaga cgttaactac 480
tttctcggaa caaactgccc tcctgttcgt gttaatggga atggaggcct tccaggagga 540
aaggtgaagc tctctggatc aattagtagt caatacttga ctgctttgct tatggcagct 600
cccttggctc taggagatgt ggagattgag attattgata aacttatttc cattccttat 660
gttgaaatga ccttgaaatt gatggaacgt tttggggtta gtgtagagca cagtcataca 720
tgggaccgat tcttgatccg aggaggtcaa aaatacaagt ctcctggaaa tgcttttgtt 780
gagggtgatg cttctagtgc tagttacttc ctagccggtg cagctgtaac tggtgggact 840
gtcacagttg aaggctgtgg gacaagcagc ctacaggggg atgtaaaatt tgctgaggtt 900
cttgagcaaa tgggtgcaaa agtttcctgg atggagaaca gtgtcacagt cacaggccca 960
ccccgagatt cttctggaag gaaacactta cgtgccattg acgtcaacat gaacaagatg 1020
ccagatgctg ccatgactct tgctgtagtt gccctttatg ctgatgggcc gactgccatc 1080
agagatgtgg ctagttggag agtgaaggag accgaaagga tgattgccat ttgcacagaa 1140
ctcaggaagc tgggagcaac agttgaagaa gggcctgatt actgtgtgat cactccaccc 1200
gaaaaactaa acgtgacatc aatagacaca tacgacgatc acaggatggc catggcattt 1260
tctcttgctg cctgtgcaga tgttccagta accatcaagg atcctggttg cacccggaag 1320
accttccctg actacttcga agtcctccag cgttttacca aacattga 1368
Claims (4)
1. one kind derive from grape (
vitis vinifera) EPSP synthase multisite mutant, it is characterized in that, described multisite mutant contains following 7 mutational sites:
Mutational site 1:Q93R, the glutamine namely in EPSP synthase aminoacid sequence on the 93rd is replaced by arginine;
Mutational site 2:T113A, the Threonine namely on the 113rd is replaced by L-Ala;
Mutational site 3:P117L, namely the proline(Pro) of the 117th replaces with leucine;
Mutational site 4:G126A, the glycine namely on the 126th is replaced by L-Ala;
Mutational site 5:C160Y, the halfcystine namely on the 160th is replaced by tyrosine;
Mutational site 6:N239H, the l-asparagine namely on the 239th is replaced by Histidine;
Mutational site 7:V343A, the α-amino-isovaleric acid namely on the 343rd is replaced by L-Ala.
2. coding according to claim 1 derive from grape (
vitis vinifera) EPSP synthase multisite mutant, it is characterized in that, the aminoacid sequence of described multisite mutant is as shown in SEQ ID No.1.
3. coding according to claim 1 derive from grape (
vitis vinifera) EPSP synthase multisite mutant, it is characterized in that, the base sequence of described multisite mutant is as shown in SEQ ID No.2.
4. the coding described in Claims 2 or 3 derive from grape (
vitis vinifera) EPSP synthase multisite mutant gene resistance glyphosate transgenic paddy rice cultivate in application.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2017128301A1 (en) * | 2016-01-29 | 2017-08-03 | 四川天豫兴禾生物科技有限公司 | Glyphosate-tolerant epsps gene screening method and use thereof |
| WO2018120707A1 (en) * | 2016-12-28 | 2018-07-05 | 四川天豫兴禾生物科技有限公司 | Paddy rice epsps mutant, and encoding gene and use thereof |
| CN108291236A (en) * | 2015-09-30 | 2018-07-17 | 先锋国际良种公司 | Plant EPSP synthase and application method |
| CN111394368A (en) * | 2020-04-29 | 2020-07-10 | 海南大学 | Hevea brasiliensis EPSPS gene with 182 th site mutation and application thereof |
| WO2022160145A1 (en) * | 2021-01-28 | 2022-08-04 | Cropedit Biotechnology Inc. | Epsps mutants and method of its uses |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103205404A (en) * | 2013-03-26 | 2013-07-17 | 上海市农业科学院 | EPSP (5-enolpyruvyl shikimate-3-phosphate) synthase multisite mutant from Malus domestica, and coding gene and application of mutant |
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103205404A (en) * | 2013-03-26 | 2013-07-17 | 上海市农业科学院 | EPSP (5-enolpyruvyl shikimate-3-phosphate) synthase multisite mutant from Malus domestica, and coding gene and application of mutant |
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| Title |
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| DA SILVA C ET AL.: "NCBI Reference Sequence: NP_001268176.1", 《GENBANK》 * |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108291236A (en) * | 2015-09-30 | 2018-07-17 | 先锋国际良种公司 | Plant EPSP synthase and application method |
| WO2017128301A1 (en) * | 2016-01-29 | 2017-08-03 | 四川天豫兴禾生物科技有限公司 | Glyphosate-tolerant epsps gene screening method and use thereof |
| WO2018120707A1 (en) * | 2016-12-28 | 2018-07-05 | 四川天豫兴禾生物科技有限公司 | Paddy rice epsps mutant, and encoding gene and use thereof |
| US10920201B2 (en) | 2016-12-28 | 2021-02-16 | Gevoto Llc | Rice EPSPS mutant, encoding gene and use thereof |
| CN111394368A (en) * | 2020-04-29 | 2020-07-10 | 海南大学 | Hevea brasiliensis EPSPS gene with 182 th site mutation and application thereof |
| CN111394368B (en) * | 2020-04-29 | 2023-04-18 | 海南大学 | Hevea brasiliensis EPSPS gene with 182 th site mutation and application thereof |
| WO2022160145A1 (en) * | 2021-01-28 | 2022-08-04 | Cropedit Biotechnology Inc. | Epsps mutants and method of its uses |
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